Pseudanabaena sp. NIES-4403 , that forms scattered (discrete) migrating colonies on solid media. While the scattered colony pattern has been observed in some bacterial species, the mechanism underlying such a pattern still remains obscure.Bacteria have been reported to exhibit complicated morphological colony patterns on solid media, depending on intracellular, and extracellular factors such as motility, cell propagation, and cell-cell interaction. We isolated the filamentous cyanobacterium, Pseudanabaena migrating collectively and found that this species forms randomly scattered clusters varying in size and further consists of a mixture of comet-like wandering clusters and disk-like rotating clusters. Quantitative analysis of the formation of these wandering and rotating clusters showed that bacterial filaments tend to follow trajectories of previously migrating filaments at velocities that are dependent on filament length. Collisions between filaments occurred without crossing paths, which enhanced their nematic alignments, giving rise to bundle-like colonies. As cells increased and bundles aggregated, comet-like wandering clusters developed. The direction and velocity of the movement of cells in comet-like wandering clusters were highly coordinated. When the wandering clusters entered into a circular orbit, they turned into rotating clusters, maintaining a more stable location. Disk-like rotating clusters may rotate for days, and the speed of cells within a rotating cluster increases from the center to the outmost part of the cluster. Using a mathematical modeling with simplified assumption we reproduced some features of the scattered pattern including migrating clusters.We studied the morphology of Pseudanabaena forms scattered migrating colonies that undergo a series of transitions involving several morphological patterns. A simplified model is able to reproduce some features of the observed migrating clusters.Based on these observations, we propose that The online version contains supplementary material available at 10.1186/s12866-021-02183-5. Bacillus subtilis colonies demonstrated that colony morphology depends on the solidity and nutrient concentrations of the media [Bacterial colonies are formed through biological self-organization –5. For ehe media , 7. Theshe media , 8.Bacillus and Paenibacillus species. For example, Paenibacillus vortex forms both wandering and rotating clusters when cells elongate in the presence of mitomycin C [Escherichia coli [Paenibacillus Alvei also forms wandering colonies [Paenibacillus sp. NAIST15–1 forms both wandering and rotating colonies [Myxococcus xanthus is a bacterium that forms large moving clusters such as vortices, bundled circular patterns, side-by-side clusters, and rafts [Self-propelled bacteria often show complicated collective behaviors, such as the formation of dense moving clusters, which is exemplified by “wandering” (comet-like) and “rotating” colonies as described by Henriksen –11. Thesomycin C  or when hia coli . Paenibacolonies , while Pcolonies . Meanwhind rafts . In thisnd rafts , 18 and nd rafts  and its nd rafts , 20 haveBacillus and Paenibacillus [Synechocystis sp. PCC 6803, which form finger-like migrating cluster toward light source [Phormidium sp. KS shows spiral vortices on agar plates [Pseudanabaena species isolated from a brackish mudflat in California was reported to show reticulate morphology in population during slurry aggregation [Pseudanabaena species (Pseudanabaena geleata OL-75-Ps) the comet-like wandering cluster is documented in a brief taxonomic catalog on the Bergey’s manual [Motile cell aggregates have also been observed in cyanobacterial species which lack flagella, an appendage used in collective behaviors of bacillus , 22. Most source –26. Evenr plates . Pseudanregation . More ins manual , while tPseudanabaena sp. NIES-4403, a filamentous cyanobacterium that forms both comet-like wandering aggregates and rotating clusters on solid media. At the macroscopic level, the mixture of wandering, and rotating clusters forms randomly scattered clusters that vary in size. The randomly scattered pattern on solid media looks similar to the “stellar” pattern that has been briefly described in Paenibacillus Alvei [Here, we report the isolation of us Alvei , while iPseudanabaena . On BG-11 solid medium, this cyanobacterium was observed to develop randomly scattered clusters that varied in size  may be rejected if the p-value is less than 0.1. On the one hand, the p-values calculated to determine the degree of fit to the power-law distribution were 0.15 and 0.01 for the experimental data at hours 100 and 200, respectively. On the other hand, the corresponding p-values determining fit to the log-normal distribution  is tested against the alternative hypothesis H1 (one of the test distributions is closer to the true distribution). A p-value greater than 0.1 indicates that it is difficult to determine which distribution is more appropriate, according to Clauset et al. [p-values for the data at hours 100 and 200 are 1.02E-08 and 1.34E-03, respectively. Taken together, our results suggest that the power-law distribution is a better fit for the experimental data at hour 100, while the log-normal distribution is a better fit for the data at hour 200. This is consistent with our findings for the CDFs for data at hours 100 and 200, in which the former follows a straighter line on a log-log plot, which indicates a power-law distribution  of cluster size distributions (including comet-like and disk-like clusters) follows a roughly straight line on a log-log plot, regardless of time after inoculation  are proposed to activate motility , 33, 34.ndia ink –38. Althnt level , while wBacillus subtilis has formed finger-shaped extending branches in which cells at the outermost tip wall remain immotile, while the inner cells randomly swirl, pushing the tip wall cells to extend the branch (division of labor). The tip wall of the comet-like wandering cluster in Pseudanabaena is also covered by layers of filaments that can be pushed by inner filaments that are aligned along the axis of the cluster. However, unlike Bacillus subtilis, the relative positions between the tip wall filaments and the inner nematically aligned filaments change little as the cluster moves  = ωR, where R is the distance of the point from the center and ω is angular speed of the rotating cluster. Although the distribution of velocity shown in Fig. n = 30) was analyzed to be compared with that of migrating clusters on agar plates  surface. When we examined the orientation of 10 random rotating clusters on each of three plates, the ratio of counterclockwise to clockwise was 6:4, 7:3, and 4:6, respectively, for a total of 56.7% clockwise and 43.3% counterclockwise. Even when comparing the two neighboring rotating clusters, some pairs rotated in the same direction, while the other pairs were in different directions. Basically, the rotational direction seems not change from the beginning of the disk-like rotating cluster formation. Change in the direction was only exceptionally observed when the rotating cluster was subjected to large perturbation by merging of another wandering cluster. Therefore, the direction of rotation is primarily defined by the bending direction of a wandering cluster when it bends into a circular trajectory for the formation of a rotating clusters. The direction in which the wandering cluster bends seems not particularly directional, and is considered to be primarily random.Pseudanabaena cells secrete EPS, although more detailed analysis is necessary to determine if it is involved in trail formation. When two filaments collide, in most cases, the filaments do not cross paths, but instead align with each other to form a bundle  and on the top line (θout≃90) indicates that colliding filaments resulted in parallel alignment and antiparallel alignment, respectively. This characteristic, also known as “nematic alignment,” has been observed in Myxococcus xanthus [Pseudanabaena as well. It should be noted that in high-density clusters, neighboring filaments are essentially aligned with each other.Except for the comet-like wandering clusters and rotating clusters, most bacterial filaments move alone or form bundles Fig. a. A bunddle Fig. . Figure  xanthus , and thiPhormidium sp. [The velocity of a single filament gliding on a solid surface without following trails  ranged from 0.02 to 0.19 μm/s, depending on the filament length Fig. e. Below dium sp. , which iPseudanabaena remains unknown. However, several lines of studies have strongly suggested that gliding of filamentous cyanobacteria is driven by a polysaccharide secretion system known as the junctional pore complex (JPC). In differentiated motile hormogonia in Nostoc punctiforme, JPC is formed with arrayed ring structure of type IV-pilus-like systems, which are encoded in part by pil and hps genes [pil and hps genes are conserved in essentially all filamentous cyanobacteria, as are several other genes that are important for motility in N. punctiforme, including those coding for the hmp chemotaxis-like system[pil and hps genes are reported to be conserved in another Pseudanabaena strain, sp. PCC 7367 [N. punctiforme appearance of dense rafts with nematic alignment is often observed, while they do not typically separate from the inoculum site [hmpW mutant [Phormidium species which forms vortex clusters [Pseudanabaena is also based on similar gliding mechanism. Nevertheless, to explain scattered high density cluster patterns exclusively observed in Pseudanabaena, further understanding on additional aspects like cell-cell interactions and physical property of filaments must be necessary.The mechanism of gliding motility in ps genes , 42–44. ke system, 43, 44 W mutant  under phclusters , 36. ThuDuring the course of cultivation, clusters may change their patterns to any of those previously described . When the density of cells/filaments is determined to be relatively low (early after inoculation), single filaments may collide, and align into a bundle as mentioned above. Figure We propose that the formation of a high-density cluster requires a positive feedback system in order to facilitate aggregation. Comet-like wandering colonies become larger Fig. b–d via bPseudanabaena filaments tend to move with nematic alignment  the direction of movement of each particle attains uniformity by nematic order; and (ii) the rotation rate of particles is maintained for a relatively long period. We wanted to ask to what extent we could reproduce the scattered pattern of Pseudanabaena with this model with some modifications. It is important to note that in this study, we are trying to reproduce at somewhat over-simplified level whether scattered colonies, comet-like wandering colonies, and rotating colonies can occur even when based on minimalist assumptions. The previous model applied for C. elegans was run in a torus field to reproduce a small space, and resulted in the formation of a dynamical network and the stabilization of particle movements. By contrast, we ran our modified model in infinite space with locally condensed particles at the initial position to represent the actual inoculation situation  or repulsive force r < rr) from neighbor particles. Parameters used in our simulation are listed in Table entclass1pt{minimaNi in order to avoid excessive increments of attraction under high-density conditions. We employed the newly fitted parameters as summarized in Table Pseudanabaena  trail following of filaments possibly through polysaccharide secretion and/or groove formation on the solid surface, (2) bundle formation with nematic alignments, (3) top covering-induced formation of comet-like wandering clusters, (4) formation of disk-like rotating clusters through spontaneous self-following of the wandering cluster, and (5) collision-based transition among different cluster types. Based on the simple assumptions of nematic interaction and temporal maintenance of the direction of motion, our agent-based model reproduced some characteristics of the scattered colony formation. Although the observed morphologies were observed under artificial conditions, we speculate that these features contribute to the adaptive fitness of this species in its natural environment. For example, the formation of a highly dense cluster without long-distance migration, as exemplified by the disk-like rotating cluster, should be of advantage in forming a stable biofilm under preferred growth conditions. In addition, the ability to coordinate migration via a comet-like wandering cluster moving at maximal speed should facilitate colony expansion or escape from undesirable condition. In other words, this cyanobacterial species has developed its own strategy to form biofilms that include some positive feedback-based aggregation and dispersing processes. The morphological patterns produced under pure culture conditions on an artificially flat solid surface are manifestations of a strategy that has evolved under natural conditions. Needless to say, natural environment is much more complicated and thus formation and characteristics of migrating clusters must be influenced by external stimuli such as light and chemoattractors. It should be noted that at the beginning of isolation of the strain in 2007 the strain had shown positive phototaxis toward the light source, while during successive inoculation at the lab it turned to show weak negative phototaxis . Cells were cultured on BG-11 medium [− 2 s− 1) at 30 °C. Subculturing was performed every 2 weeks by inoculating 4 μl of cell suspension on fresh BG-11 agar plates.1 medium  containiE. coli JM109 cells . The PCR-derived segments of the resulting plasmids were sequenced  using the same primers, and the resulting sequence was compared to 16S rRNA gene sequences from 29 cyanobacterial species  on Mar 23, 2018) [Synechocystis sp. PCC 6803, Nostoc punctiforme PCC 73102, Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, and Synechococcus elongatus PCC 7942) were chosen to compare with the sequence derived from Pseudanabaena. These 16S rRNA gene sequences and that of Pseudanabaena sp. were aligned with MUSCLE (version 3.8.31) [A region of the 16S rRNA gene was then amplified by colony PCR using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1494R (5′-GTACGGCTACCTTGTTACGAC-3′). The amplified DNA fragment was further cloned into the pGEM-T Easy vector  and transformed into 3, 2018) . Twenty-3, 2018) , excludi 3.8.31) , calcula 3.8.31) , and use 3.8.31)   for both photography and cell culture. A thermo glass plate  set to 33 °C was placed on top of the agar plates to maintain the growth temperature and avoid condensation. For any microscopic observations except for Fig. − 2 s− 1 from the bottom or the upper side to avoid directional effect which may cause phototactic motility as much as possible. For experiment shown in Fig. Microscopic images were taken using a CCD camera  coupled to an inverted microscope  equipped with UPlanFLN (4x) and LUCPlanFLN (20x) objectives (both Olympus). The system was controlled by SlideBook software . Confocal microscopic images were taken using the FV-1000D system (Olympus) connected to an automated microscope  equipped with the LUCPlanFLN (20x) objective (Olympus) and controlled by FV10-ASW software. Five hundred fifty-nine nanometre laser excitation was used to visualize the autofluorescence from cyanobacterial filaments. For confocal imaging, cells were cultured on 2- ml of BG-11 solid media containing 1.5% of Gelrite  instead of agar for clarity, on 35-mm plates with glass bottom . We confirmed that the colony patterns produced by Images were processed and analyzed with ImageJ 1.50b   and FijiFor passing count images shown in Figs. The values of actual velocity of each cluster shown in Figs. The values of actual velocity of filaments shown in Fig. For PIV analysis shown in Figs. Θin is the incoming angle between the lines with 10-pixel long from each colliding filament to the collision point, while θout is the angle between the lines from colliding point to 10-pixel forward to the moving direction of each filament using the line selection tool in Fiji.Angle of colliding filaments shown in Fig. KSd) of the estimated and the experimental datasets. Then, we performed a goodness-of-fit test. Then, a dataset of composite values was synthesized from the estimated equation. This dataset was prepared so that the ratio of the number of data points above the cut-off value to that below the cut-off value was equivalent to that of the experimental data. For this synthetic dataset, data above the cut-off value were randomly selected to follow the estimated distribution, while data below the cut-off value were randomly sampled from the experimental data below the cut-off value. For each synthetic dataset, the KS value (KSsim) was calculated using the same steps as those performed to calculate KSd. This process was repeated, following the bootstrapping method, enabling us to determine the probability (p) of KSd appearing greater than KSsim. Clauset et al. [H0  should be rejected if the p-value is less than 0.1. To select the model distribution, we performed the Vuong’s test, which is a likelihood ratio test for model selection using the Kullback-Leibler criterion [H0: both distributions are equally far from the true distribution versus the alternative hypothesis H1: one of the test distributions is closer to the true distribution. If the p-value given by the test is greater than 0.1, then the test is inconclusive, according to Clauset et al. [The size of any detectable colonies observed in Movie t et al.  have proriterion , 32. Thit et al. .Cells were fixed with 2.5% glutaraldehyde dissolved in 0.1 M phosphate buffer solution at 4 °C, washed with phosphate buffer solution, and additionally fixed with osmium tetroxide at 4 °C. Samples were then dehydrated several times in a 50 to 100% ethanol series and then freeze-dried. For SEM observation, dehydrated cells were coated with osmium and observed using a JSM-6320F SEM . For TEM observations, dehydrated cells were embedded in epoxy resin , and ultrathin slices of the material were doubly stained in uranyl acetate and lead citrate. The sections were coated with carbon in a vacuum vapor deposition system, and then the coated ultrathin slices of cells were observed using TEM . All observations were performed at the Hanaichi UltraStructure Research Institute .2, and 0.05% (v/v) Triton X-100 (Sigma) was then dropped on the surface of the medium. Photos were taken under the microscope (IX71) with UPlanFLN (4x) objective  before staining and 2 min after staining.For Alcian blue staining, 20 μl of cell suspension was incubated on a slide glass for 30 min. Any floating cells and liquid were removed gently by absorbing to filter paper. The materials remaining on the slide, including the cells, were stained with 20 μl of 1.5% (w/v) Alcian blue 8GX  for 30 min. Then, the Alcian blue solution was replaced by 20 μl of BG-11 liquid medium, and a coverslip was then mounted onto the droplet. Stained materials were observed under the microscope  with an UplanFLN (10x) objective , and images were collected using the Color CMOS camera . For ink staining, cells cultured on BG-11 agar medium for about 10 days were used. Twenty microlitre of solution containing 2% india ink , 4μMCaClAdditional file 1: Figure S1. Phylogenetic analysis based on the 16S RNA gene. 16S rRNA gene sequence of Pseudanabaena sp. NIES-4403 was compared to those deposited with the Ribosomal Database Project (RDP), including 24 closely related sequences chosen according to BLAST search results and 5 representative cyanobacterial strains. Numbers on the junctions represent bootstrap values.Additional file 2: Figure S2. Velocity distribution of comet-like wandering clusters and disk-like rotating clusters. The velocity of rotating clusters was measured on the circumference. Stars indicate significancy different distributions with p-value of 7.293 × 10− 9 with Wilcoxon’s rank sum test.Additional file 3: Figure S3. Trajectories and changes in the velocity and the size of three representative comet-like wandering clusters . White circles show standard distance from the center position.Additional file 7: Figure S7. Regression analysis on the filament length and moving velocity. The regression line shown in Fig. Additional file 8: Figure S8. Negative phototactic behavior of Pseudanabaena. Cell suspension was put on the center of agar-containing medium on a 90-mm plate and incubated for about 10 days. An arrowhead indicates the direction of illumination.Additional file 9: Movie S1. Time-lapse images of Pseudanabaena on a 90-mm agar plate. Cells were inoculated at the center and incubated for about 1 day before recording the video. Colors indicate the position of the cluster shown in Figs. Additional file 10: Movie S2. Time-lapse movies of comet-like wandering cluster  and a disk-like rotating cluster (lower right) are visible. Some bundled filaments and single filaments are also visible.Additional file 13: Movie S5. Time-lapse confocal images of a comet-like wandering cluster.Additional file 14: Movie S6. Time-lapse images of disk-like rotating cluster (extracted from Movie Additional file 15: Movie S7. Time-lapse images on a 90-mm agar plate (same image source for Movie Additional file 16: Movie S8. Time-lapse confocal images of a disk-like rotating cluster.Additional file 17: Movie S9. Time-lapse images of a bundle composed of aligned filaments. The bundle visible at the beginning of this movie consists of nine filaments.Additional file 18: Movie S10. Time-lapse of images of a single filament following a trail.Additional file 19: Movie S11. Collision between two filaments, moving parallel to each other.Additional file 20: Movie S12. Collision between two comet-like clusters, forming a unified cluster.Additional file 21: Movie S13. Transition from a comet-like cluster to a rotating cluster.Additional file 22: Movie S14. Collision between a comet-like cluster and a disk-like rotating cluster, leading to the collapse of the rotating cluster.Additional file 23: Movie S15. Video of the simulated result of the self-propelled particle model.Additional file 24: Movie S16. Time-lapse images of a representative wandering cluster on simulation (extracted from Movie Additional file 25: Movie S17. Time-lapse images of a representative rotating cluster on simulation (extracted from Movie Additional file 26: Dataset S1. Original dataset used for figures and tables were compiled.


Previous observational studies have suggested that associations exist between growth differentiation factor 15 (GDF-15) and neurodegenerative diseases. We aimed to investigate the causal relationships between GDF-15 and Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).p < 5 × 10–8) with GDF-15 were selected as instrumental variables (n = 5). An inverse-variance weighted method was implemented as the primary MR approach, while weighted median, MR–Egger, leave-one-out analysis, and Cochran’s Q-test were conducted as sensitivity analyses. All analyses were performed using R 3.6.1 with relevant packages.Using summary-level datasets from genome-wide association studies of European ancestry, we performed a two-sample Mendelian randomization (MR) study. Genetic variants significantly associated (p = 0.004). In the reverse direction, Mendelian randomization suggested no causal effect of genetically proxied risk of AD on circulating GDF-15 (p = 0.450). The causal effects of GDF-15 on PD (p = 0.597) or ALS (p = 0.120) were not identified, and the MR results likewise did not support the association of genetic liability to PD or ALS with genetically predicted levels of GDF-15. No evident heterogeneity or horizontal pleiotropy was revealed by multiple sensitivity analyses.MR provided evidence for the association of elevated GDF-15 levels with a higher risk of AD (odds ratio = 1.14; 95% confidence interval, 1.04–1.24; We highlighted the role of GDF-15 in AD as altogether a promising diagnostic marker and a therapeutic target. Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) are common neurodegenerative diseases of aging populations, which lead to the growing healthcare burden worldwide. Most clinical trials targeting potential biochemical pathways in AD, PD, and ALS or modifiable lifestyle factors have failed, leaving merely a handful of medications to ease patient suffering partially or transiently so far . ResearcGrowth differentiation factor 15 (GDF-15) belongs to the transforming growth factor-β superfamily . GDF-15 p = 0.03), but it should be noted that prior smaller cohorts were prone to confounding bias , and validation of their findings in a larger sample size is required. Studies exploring the relationship between GDF-15 and PD  have been reported (Mendelian randomization (MR) is emergingly utilized as an efficient tool to explore the causal relationships between a wide spectrum of exposures, including serum biomarkers, and diseases of interest . MR is nreported . These Sreported , PD . The other three cohorts were comprised of randomly recruited community-dwelling participants (∼900 in each cohort). In total, the meta-analysis comprised 5,440 participants with a mean age of 62.1 years, among which 53.1% were female (P < 5 × 10–8) were kept as instrumental variables (Beta) was presented in a unit of SD change in GDF-15 levels per additional effect allele. In the reverse MR investigating whether there were causal effects of neurodegenerative diseases on circulating GDF-15 levels  were reported and further utilized as instrumental SNPs  as proxies for PD in the MR analysis of genetically predicted risk of PD on circulating GDF-15  to investigate the effect of genetically proxied liability to ALS on circulating GDF-15  model , with the TwoSampleMR (version 0.5.3) package  and the W) model , gave anr GDF-15  was inpur GDF-15  to generr GDF-15 , and henr GDF-15 . MR–Egger GDF-15 , whereasvariants . A postevariants  at the gp = 0.004) per one SD increment in genetically predicted concentrations of GDF-15. The weighted median estimator also provided suggestive evidence for the relationship  but failed to reach Bonferroni significance. Notably, Cochran’s Q-test  and MR–Egger regression  indicated no existence of pleiotropic or outlying variant which would drive the overall estimate disproportionately. In this case, weighted median and MR–Egger regression were not likely to generate as precise estimates as the IVW approach. According to our power estimation, given the variance of GDF-15 explained by instrumental variables of 21.5%, the MR analysis has sufficient power (> 80%) to detect a large effect size (OR > 1.057 or OR < 0.945). Although no heterogenous variants were revealed in the scatter plot (p = 0.007) and rs1227731  were notably nominally significant (p < 0.05) when examining the effect of GDF-15 on AD mediated by individual SNP , and rs1227731 (chr19:18497903) was located in the intron between the only two exons of GDF15 . Albeit in non-coding regions, given the proximity and the specificity of their locations  by the IVW method. Scatter plots (p = 0.202) and Cochran’s Q-test  indicated no existence of horizontal pleiotropy or obvious heterogeneity.In the reverse direction, MR analyses  did not er plots  and leaver plots  suggestep = 0.597). There was likewise no causal effect of GDF-15 on the risk of ALS  per one-SD increase in GDF-15 levels. Additional MR methods provided consistent results. Furthermore, no heterogeneity was identified in the MR analysis on PD  or ALS , and no pleiotropy was detected for PD  or ALS . Nevertheless, this study would be underpowered to detect the OR intervals of 0.966–1.034 for PD and 0.952–1.049 for ALS.The MR analyses did not support the relationship between GDF-15 and PD or ALS . A changp = 0.062) per one-unit increase in log-OR of PD, whereas it was −0.002 SD  for ALS. Multiple sensitivity analyses , implicating its predictive value as a novel AD biomarker. Another recent study . TGFβRII (TGFβ receptor type II), which is mainly expressed in microglia and neurons, serves as the mediator and receptor and bridges the effect of GDF-15 on IDE. IDE is required downstream for Aβ degradation in neurons and microglial cells  in a Japanese cohort  in ALS patients. One recent study (CHCHD10 in ALS demonstrated that GDF-15 was involved in the activation of the mitochondrial unfolded protein response, which might contribute to the selective vulnerability of motor neurons in the pathogenesis of ALS. More studies are necessary to elucidate the relationship between GDF-15 and PD and ALS.There have not been enough studies  explorinnt study  investigThe primary strength of this study lies in the application of two-sample MR, which has benefits in circumventing residual confounding, avoiding reverse causation, and enhancing power by a large sample size. There are several limitations as well. Firstly, genetic variant-proxied GDF-15 levels represent overall serum levels across the lifespan. We should be cautious with the effect estimate in regards to translational medicine and pharmaceutical development, even if GDF-15 has the potential to be a target, since drug treatment generally takes effect in a smooth and transient fashion. The finding was also based on European-ancestry datasets, and the participants in the GWAS of GDF-15 were from four community-based cohorts, whereas the participants in the GWAS of AD were from a much larger population; great caution should be exercised in the interpretation and generalization.In conclusion, we highlighted the role of GDF-15 in AD as a promising diagnostic marker and therapeutic target. Triangulating evidence across clinical observational, genetic epidemiological, and biological mechanistic studies is warranted to shed light on whether targeting GDF-15 is an effective treatment for AD, PD, and ALS.The original contributions presented in the study are included in the article/P-FW, X-HZ, and PZ contributed to the conception and design of the study. P-FW, X-HZ, RY, and X-TZ contributed to the acquisition and analysis of data. P-FW, X-HZ, and WZ contributed to the drafting and review of the main manuscript, tables, and figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aims of this study were to assess whether there is a conceptual overlap between the questionnaires HIT-6 and EQ-5D and to develop a mapping algorithm allowing the conversion of HIT-6 to EQ-5D utility scores for Germany.This study used data from an ongoing randomised controlled trial for patients suffering from migraine. We assessed the conceptual overlap between the two instruments with correlation matrices and exploratory factor analysis. Linear regression, tobit, mixture, and two-part models were used for mapping, accounting for repeated measurements, tenfold cross-validation was conducted to validate the models.We included 1010 observations from 410 patients. The EQ-5D showed a substantial ceiling effect (47.3% had the highest score) but no floor effect, while the HIT-6 showed a very small ceiling effect (0.5%). The correlation between the instruments’ total scores was moderate (− 0.30), and low to moderate among each domain (0.021–0.227). The exploratory factor analysis showed insufficient conceptual overlap between the instruments, as they load on different factors. Thus, there is reason to believe that the instruments’ domains do not capture the same latent constructs. To facilitate future mapping, we provide coefficients and a variance–covariance matrix for the preferred model, a two-part model with the total HIT-6 score as the explanatory variable.This study showed that the German EQ-5D and the HIT-6 lack the conceptual overlap needed for appropriate mapping. Thus, the estimated mapping algorithms should only be used as a last resort for estimating utilities to be employed in economic evaluations.The online version contains supplementary material available at 10.1007/s10198-021-01342-9. Migraine is a common neurological condition affecting 10.6% of the German population  , 3.This condition also imposes an economic burden on health care systems due to increased demand for goods and services and work-related productivity losses , 5. As hClinical trials in migraine often use monthly migraine days as a primary endpoint and the International Headache Society actually recommends the use of monthly migraine days as the primary endpoint for HTAs involving preventive treatments . Where gNevertheless, several trials in the migraine field e.g. , 10) onl onl10]) One study by Gillard et al. has already mapped the EQ-5D to the HIT-6, but used quality weights for England in a Brazilian population . SeveralBased on these considerations, we will address the issue of whether there is enough conceptual overlap between the two instruments using not only correlation tables, but also exploratory factor analysis (EFA). Based on regression-type approaches we will develop a mapping function to predict EQ-5D utility values for Germany from HIT-6 values, including variables widely used in migraine trials, and validate them with tenfold cross-validation.This study is based on data from the SMARTGEM project. SMARTGEM is an ongoing national randomised controlled clinical trial, which seeks to assess if a digital intervention via the use of a headache app and online consultations leads to a decrease in migraine frequency. The intervention consists of a certified medical app where patients document trigger factors, attacks, and medication in an electronic calendar; the app analyses the diary and evaluates trigger factors and proposes individually tailored treatment plans; a web-based tool where patients communicate both with other patients and specialists. HIT-6 and EQ-5D were completed by all users at baseline and after 3, 6, 9, and 12 months. Registration ID in the German clinical trials register is DRKS00016328.To analyse the strength of the relationship between HIT-6 scores and EQ-5D domains, correlation coefficients accounting for repeated measurements were computed. We also examined the capacity of each instrument to detect changes in HRQOL over time, referred to as responsiveness, by computing standardised response mean(s) (SRM). SRM is defined as a ratio of the difference in the mean baseline and mean follow-up values divided by their mean standard deviations’ (sd) difference. We considered SMR values of less than 0.2 as small, from 0.2 to 0.5 as moderate, and values above 0.8 as large, following Cohen’s criteria . EFA wasSince there is no specific model recommended by guidelines on best practices for mapping, we applied several models . As our We also fitted adjusted limited dependent variable mixture models with one to four components, with the Stata command aldvmm, which was specifically developed to deal with health utility data . These mHIT-6 related variables, sex, age, and migraine type were pre-defined as having to be part of the model. Given that mapping algorithms are intended to be used by other researchers, we only considered age and sexWe conducted complete-case analysis based on the following variables: EQ-5D domains, HIT-6 domains, migraine type, age, and sex.R2, reporting the mean of all 10 cycles.We plotted the observed and predicted EQ-5D values to visualise the models’ performance. Given the lack of external data to conduct external validation, a tenfold cross-validation was carried out to compare the predictions of each model with the actual EQ-5D scores. This method is recommended for small samples . Models’R 3.6.3 and Stata 15 [R packages for data handling [Statistical analyses were performed using Stata 15 , 34. We handling  and plothandling , repeatehandling , and fachandling , 39, a Shandling   observations had to be removed. thus the dataset used for the analysis contains 1010 observations, based on 410 patients. 7 out of 16 were excluded from the analysis because they did not have full data on other time points.87.3% of all participants were female, with an average age of 41 years . The skewness of − 0.64 indicated that the HIT-6 scores are only slightly skewed to the left Fig. . There wIn EQ-5D, there was no floor effect , i.e. no patient had the lowest utility score possible . However, the ceiling effect  amounted to 47.3% (194/410).We consider that occupation and daily activities can be measured by the EQ-5D dimension “usual activities” and by questions 2, 3, and 4 from the HIT-6. Physical health is captured by “pain/discomfort” and “mobility” in the EQ-5D, and by question 5 from the HIT-6. Self-care is only measured by the EQ-5D.The correlation coefficient between EQ-5D score value and the HIT-6 total score amounted to − 0.30. In terms of EQ-5D value and the different HIT-6 dimensions, the coefficients ranged between − 0.153 and − 0.234. The correlation coefficients between each EQ-5D domain and the overall HIT-6 score ranged from 0.077 to 0.300 (Table A.1). Lastly, the correlation coefficients among each domain from the two instruments ranged from 0.021 to 0.227. The highest correlation (0.227) was found between EQ-5D pain/discomfort and HIT-6 q4. See Supplementary Tables A.1, A.2, and A.3 for correlation tables, additionally stratified by migraine severity level.The EQ-5D total score and the different dimensions show small SRMs, while the HIT-6 total score and its different questions show small to moderate responsiveness. For EQ-5D dimensions, SRM values range from 0.088 to 0.280 and for the HIT-6 from 0.211 to 0.669 (see Supplementary Table A.4). Although the lack of responsiveness may be in part because we are also analysing patients in the control group, this still does not explain why the responsiveness of the HIT-6 is higher than that of the EQ-5D.We considered three factors in the EFA. Factor 1 had meaningful loadings (i.e. higher than 0.3) on all EQ-5D domains, but not on HIT-6 domains. Factors 2 and 3 loaded only on HIT-6 domains, specifically questions 2–6 for Factor 2, questions 1 and 2 for Factor 3. Considering that this question had a higher loading in Factor 2, thus belonging to this factor, Factor 2 had meaningful loadings in five out of six HIT-6 domains  with the state directly after full health (0.974) may explain why these models did not perform better.No model performed best across all goodness-of-fit measures. Model E  performed the best in terms of RMSE Table . AlthougHence, if researchers wish to estimate utilities from the HIT-6 to be employed in cost-utility analyses, Model E should be the preferred model. The corresponding variance–covariance matrix is available in the Electronic Supplementary Material, in Table A.6, to allow probabilistic sensitivity analysis to be carried out and account for uncertainty. However, we would like to remark that this mapping algorithm should only be used as a last resort.We aimed to assess whether there is a conceptual overlap between the HIT-6 and the EQ-5D and to present a mapping algorithm for the estimation of the EQ-5D score (with German weights) from the HIT-6 questionnaire, a disease-specific survey widely used in clinical trials with migraine patients. Our study points to major differences in the underlying constructs of the HIT-6 and the EQ-5D. The EQ-5D showed a high ceiling effect and small SRMs across time, whereas the HIT-6 did not show a ceiling effect and had a higher responsiveness. This study also provides a mapping algorithm which can be used to map HIT-6 values to EQ-5D utility values.We expected some overlap between the two instruments since both have been validated in migraine patients. The strength of association between the instruments measured with correlation coefficients was only low to moderate—both for the total scores and for each instrument’s individual questions. Furthermore, the EFA showed that the HIT-6 and EQ-5D do not have a sufficient conceptual overlap and potentially estimate different underlying constructs. There are several reasons that might explain the lack of overlap. First, the recall period in the instruments’ questions is different. While all EQ-5D questions refer to the day the questionnaire is filled out, three questions in the HIT-6 refer to the previous 4 weeks. Second, the HIT-6 has frequency response categories , while the EQ-5D has response categories based on levels of severity. Third, the specificities of both the EQ-5D and the HIT-6 may also play a role. A criticism of the use of the EQ-5D to describe health utilities in patients with migraine is the fact that the survey is conducted at random points in time, thus not differentiating whether or not patients were having a migraine attack at the moment they filled out the survey . The 47.Given the lack of responsiveness, as well as the substantial ceiling effect of the EQ-5D for migraine patients, economic evaluations with these patients should consider other approaches to determine value, not necessarily QALYs obtained from generic utility-based instruments. In fact, the guidelines of the International Headache Society state that QALYs may fail to account for specific patient preferences due to the insensitive nature of utility instruments . Thus, tStrengths of our study include the fact that trained migraine neurologists provided the migraine diagnosis to the study participants’ and the low percentage of missing data. Furthermore, we could use multiple observations per person and evaluated this data with methods suitable for repeated measurements where possible. The conceptual overlap of EQ-5D and HIT-6 was evaluated carefully prior to investigating mapping algorithms, where the latter were carried out with a broad set of multivariable modelling approaches.A limitation of our study is that no external validation could be carried out since no dataset containing both EQ-5D answers and HIT-6 was available. Randomised controlled trials are often considered the ‘gold standard’ for evidence-based medicine , and altOur results suggest that the German versions of EQ-5D and the HIT-6 are not measuring the same underlying concepts due to conceptual differences. Therefore, mapping algorithms shall only be used as a last resort for estimating utilities to be employed in cost-utility analyses.Supplementary file1 (XLSX 16 KB)Supplementary file2 (DOCX 29 KB)Below is the link to the electronic supplementary material.


EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival.The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients’ characteristics.One hundred and seventy-seven patients with advanced, EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS.Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC.A tail plateau was found in the survival curves of patients with advanced, The online version contains supplementary material available at 10.1186/s12885-022-09421-7. Globally, lung cancer cases and deaths are increasing. In 2020, GLOBOCAN estimated 2.2 million new cases  and 1.79 million deaths , making it the most frequent cancer and cause of death due to cancer .EGFR) gene mutations were discovered in 2004 and it was reported that EGFR tyrosine kinase inhibitors (EGFR-TKIs) shrink tumors of NSCLC carrying this mutation [EGFR-mutated NSCLC had been reported, and in all of these trials, the EGFR-TKIs groups had significantly better results in term of progression-free survival (PFS) [EGFR-mutated NSCLC as well as other EGFR-TKIs with or without anti-Vascular Endothelial Growth Factor (VEGF) antibodies [EGFR-mutated NSCLC.The epidermal growth factor receptor  –7. In thal (PFS) . Therefotibodies . In the tibodies , with thALK) is one of the receptor tyrosine kinases. The fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and the ALK gene was discovered in lung cancer cells in 2007 [ALK-rearranged NSCLC [ALK-rearranged NSCLC. OS in the ALEX trial was 57.4 months in the crizotinib group and unpredictable in the alectinib group, indicating a prominent prolongation of OS.Analplastic lymphoma kinase  0–1 and adenocarcinoma, suggesting that good PS is a factor for long-term survival in the study published in 2010 [EGFR-mutated NSCLC were five-year survivors in the study published in 2016 [EGFR-mutated NSCLC patients, and were reported several years ago with shorter follow-up periods. Immune checkpoint inhibitors (ICIs) have shown the benefit of prolonging PFS and OS with a “tail-plateau” on Kaplan Meier survival curves, and have been introduced into daily clinical practice [EGFR-mutated or ALK-rearranged NSCLC.There are not many reports showing long-term efficacy for patients with  in 2010 . Twenty  in 2016 . On multpractice . On the EGFR-mutated and ALK-rearranged NSCLC, in real-world clinical settings.The primary objective of this study was to explore the survival past five years from the initiation of chemotherapy. Secondary objective was to investigate factors that lead to long-term survival in EGFR-mutated or ALK-rearranged NSCLC, or radiotherapy with curative intent, and received their first chemotherapy at Juntendo University Hospital between December 10, 2008, and September 30, 2015. The presence of an EGFR mutations was evaluated by the Cycleave method or Scorpion-ARMS method, and ALK rearrangement was evaluated by a highly sensitive immunohistochemical (IHC) method or fluorescent in situ hybridization (FISH). The above criteria were met for 177 patients with EGFR -mutated or ALK-rearranged NSCLC; 155 patients had EGFR-mutated and 22 patients had ALK-rearranged NSCLC.Patients enrolled in this study had clinical stage III, clinical stage IV, or postoperative recurrence of th edition, 2012) [All data were retrospectively collected from electronic medical records. Patient characteristics, treatment, and outcomes were extracted. Patient-specific variables included age, sex, smoking status, Eastern Cooperative Oncology Group Performance Status Scale (ECOG PS), tumor histology, genetic mutation, staging, presence of extrathoracic metastasis, and presence of brain metastasis Tables  and 3. Sn, 2012) . All patP < 0.05. Hazard ratios were calculated with univariate and multivariate analysis   was calculated from the date of initiation of first-line chemotherapy to the date of death, and was censored at the date of the last visit for patients whose deaths could not be confirmed. Subgroup analyses of OS were performed. Descriptions of the eight subgroups are listed in Supplemental Fig. l) Table . All anaEGFR mutations and 22 with ALK rearrangements. The median follow-up time was 36.9 months  in the total cohort. The median follow-up time were 35.5 months  with EGFR mutations and 51.8 months  with ALK rearrangements. The median follow-up time was 89.2 months (64.6–131.4) in overall surviving patients. The median follow-up time were 89.4 months  with EGFR mutations and 82.8 months  with ALK rearrangements in survival patients. At the time of database lock, of the EGFR-mutated NSCLC patients, 17 patients (10.9%) were alive, 17 (10.9%) had been lost to follow up, and 121 (78.0%) had died. On the other hand, six patients (27.2%) were alive, three (13.6%) were lost to follow-up, and 13 (59.0%) had died among those patients with ALK rearrangements. Thirty-seven patients (23.8%) with EGFR mutations and nine (40.9%) with ALK rearrangements survived more than five years. In the patients with an EGFR mutation, the median total duration of EGFR-TKI treatments was 18.09  months; for cytotoxic chemotherapy, it was 8.08  months; and for immunotherapy, it was 1.34  months. In the ALK-rearranged patients, the median total duration of ALK-TKI treatment was 31.36  months; for cytotoxic chemotherapy, it was 8.17  months; and for immunotherapy, it was 0.69  months.All 177 included patients started their first chemotherapy between December 10, 2008, and September 30, 2015. There were 155 patients with The clinical and pathological characteristics of patients are shown in Table The median number of chemotherapeutic regimens was three , the median number of molecular targeted drugs was two , and the median number of cytotoxic chemotherapy was one .EGFR-mutated patients and 46.0  % in ALK-rearranged patients. Median OS was 36.9  months in EGFR-mutated patients and 55.4  months in ALK-rearranged patients       was 40.6  months, the one-year survival rate was 89  %, the three-year survival rate was 54  %, and the five-year survival rate was 28  %  with Exon 19 deletion, 63 patients (40.6%) with Exon 21 L858R, four patients (2.6%) with Exon 18 G719A/C/S, and three patients with Exon L861Q ; ECOG PS ; Stage ; brain metastasis ; extrathoracic metastasis   were alive for longer than five years. On the other hand, of 22 patients with ALK rearrangement, nine (40.9%) were alive for longer than five years.The clinical and pathological characteristics of patients surviving five years or more are shown in Table EGFR mutations who have survived more than five years since their first chemotherapy; 1–37 designates each patient, as thirty-seven patients were alive for more than five years. In five patients, disease had been stable with just one EGFR-TKI, and in two, disease had been stable with two EGFR-TKIs. In the other patients, not only EGFR-TKIs, but also cytotoxic chemotherapy or immunotherapy were used to control their disease. In cases with long-term survival, EGFR-TKIs tended to be used for a longer period than the other agents, and some cases survived for 10 years or more with longer EGFR-TKI use. Figure ALK-rearranged patients who have survived more than five years since their first chemotherapy; 1–9 designates each patient, as nine patients were alive for more than five years. Cases 1, 2, 4, and 9 were treated before the launch of ALK-TKI, and cytotoxic chemotherapy was used as first chemotherapy. As cases 7 and 8 were treated after the launch of alectinib, this was used as their first chemotherapy. In case 3, the disease was stable with a second generation ALK-TKI. In cases 5 and 9, disease was stable even after cessation of ALK-TKI.Figure EGFR-mutated NSCLC [EGFR mutation demonstrated improved disease-free survival (DFS) over standard-of-care chemotherapy, but the advantage of DFS did not translate into a significant difference in OS. IMPACT (WJOG6410L) is randomized phase 3 trial that compared gefitinib with vinorelbine plus cisplatin as an adjuvant treatment for stage II to III EGFR-mutated NSCLC after complete resection, but gefitinib cannot significantly prolong DFS and OS [EGFR-mutated NSCLC. DFS was significantly longer among those who received osimertinib than among those who received placebo, but OS data are too immature to allow evaluation of any benefit here [ALK-rearranged NSCLC.As described in the introductory section, the discovery of driver oncogenes and the development of their specific inhibitors have improved the survival of patients with advanced NSCLC. ICIs were also found to be effective for the treatment of advanced NSCLC, and as they can suppress cancer progression for a longer time, a “tail-plateau” can be seen in Kaplan–Meier OS curves. There is no evidence that molecular targeted agents have the potential to cure lung cancer or to suppress cancer progression for a long time like ICIs. In the adjuvant setting, there is a phase 3 study, CTONG 1104, that compared gefitinib with vinorelbine plus cisplatin as adjuvant treatment for stage II-IIIA (N1-N2) ed NSCLC . AdjuvanS and OS . ADAURA fit here . We coulEGFR-mutated and ALK-rearranged NSCLC. The MST of all patients was 40.6 months. In the subgroup analysis, the MST in EGFR-mutated NSCLC patients was 36.9 months and the MST in ALK-rearranged NSCLC patients was 55.4 months, tending to be longer in the latter. The five-year survival rate of EGFR-mutated NSCLC patients was 23.8%, and that of ALK-rearranged NSCLC patients was 40.9%. When multivariate analysis was performed on long-term survival factors, applying Cox’s proportional hazard model, six factors were significant. In descending order of hazard ratio these are: ECOG PS, postoperative recurrence, driver mutation, presence or absence of brain metastasis, sex, and age. And an approximately 2.7-fold difference was observed between the PS: 0–1 group and the PS: 2–4 group.In this study, we investigated factors related to long-term survival with advanced EGFR mutations and ALK rearrangements, and the survival curve appears to have a tail-plateau as shown in Supplemental Fig. ALK-rearranged NSCLC (cases 5 and 9), their disease was stable even after cessation of their ALK-TKI. This suggests an ALK-TKI for ALK-rearranged NSCLC has more potential to suppress the disease for a longer time like immunotherapy in patients at an advanced stage, and is effective for improving OS in an adjuvant setting than EGFR-TKI for EGFR-mutated NSCLC.We also investigated the clinical course of long-term survivors after five years. There are some long-term survivors among patients with EGFR- mutated inoperable or recurrent NSCLC in 2018. Since the subjects of this study were patients whose first chemotherapy was before osimertinib approval, from December 2008 to August 2015, 69% (108/155 patients) of patients received first- and second-generation EGFR-TKIs as their first chemotherapy. In this study, the OS of EGFR-mutated NSCLC was 36.9 months, which was longer than that in the IPASS study, at 21.6 months [EGFR-mutated lung cancer was 30.9 months. The five-year survival rate was 23.8%, which tended to be longer than previously reported [In Japan, osimertinib was approved and used as the first chemotherapy for 6 months . The reareported .ALK-rearranged inoperable or recurrent NSCLC in 2012. Later, a phase 3 trial was conducted comparing pemetrexed or docetaxel as second-line chemotherapy and cisplatin or carboplatin plus pemetrexed as first-line chemotherapy with crizotinib, and a significant prolongation of PFS was observed [In Japan, crizotinib was approved and used as the first chemotherapy for observed . In addiEGFR- mutated advanced NSCLC treated with an EGFR-TKI and identified tumor burden, quantified by the number of metastatic sites, as one such feature [When multivariate analysis was performed on long-term survival factors, applying Cox’s proportional hazard model, six factors  were significant. Park et al. and Lee et al. examined clinical features associated with survival in patients with  feature , 24, 25. feature .This research, however, is subject to several limitations. The first is that owing to the retrospective design, undefined biases may have been present and influenced the clinical outcome. The next is that the data collection and analysis were performed at a single center, and the patient sample size is small. Finally, the patients chosen for this study were those in whom initial treatment was started more than five years ago, and the standard treatment at the time was different from current therapy.EGFR mutations and ALK rearrangements, and the survival curve appears to have a tail-plateau. However, most patients were treated with several types of chemotherapy, and were on treatment at the last follow up. In two NSCLC patients with ALK rearrangement, their disease was stable even after cessation of ALK-TKI, suggesting that ALK-TKI for ALK-rearranged NSCLC has more potential to suppress the disease for a longer time like immunotherapy in patients with advanced stage disease, and is effective at improving OS in the adjuvant setting than EGFR-TKI for EGFR-mutated NSCLC.There are long-term survivors among patients with Additional file 1:Supplemental Table 1. Distribution of EGFR mutation.Additional file 2.


Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking a large N-terminal domain. Both dArc isoforms are related to the Ty3/gypsy retrotransposon capsid, consisting of N- and C-terminal lobes. Structures of dArc1, as well as capsids formed by both dArc isoforms, have been recently determined. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. A truncated N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids.  Memory formation in the brain is dependent on synaptic plasticity, and the activity-regulated cytoskeleton-associated protein (Arc) plays an important role in this process , 2. Arc Arc forms capsid-like structures that may transfer information from one neuron to another , 16. MamDrosophila has two Arc isoforms (dArc1 and dArc2), which share high sequence similarity. Drosophila Arc (dArc) isoforms have a CT domain, containing tandem N-and C-lobes, but lack an Arc-NT found in mArc. However, dArc forms capsids [ capsids , whose s capsids . In addi capsids . Whether capsids , but theWe set out to determine structures of the individual dArc lobe domains. The CL of both dArc1 and dArc2 is structurally homologous to the mArc-CL, confirming the connection of dArc to both mArc and retroviral capsids. The structure of dArc2-NL showed a domain-swapped dimer, resulting in a structure similar to the flavivirus capsid protein and resembling histones as well as membrane-interacting BAK domains. All individual dArc lobes were oligomeric in solution, in contrast to the monomeric mArc-CT. Such oligomeric units, reflective of the different evolutionary pathways leading to mammalian and insect Arc, could be building blocks during the assembly of virus-like capsids by full-length dArc, or they could relate to other functions of dArc.Escherichia coli BL21(DE3) with a TEV protease-cleavable His tag-maltose binding protein (MBP) fusion at the N terminus. Cells were grown at +37°C until an A600 of 0.6 was reached. 1 mM isopropyl β-D-1-thiogalactopyranoside was added to start the induction, lasting 4 h at +30°C. The cells were lysed in HBS  containing 0.1 mg/ml lysozyme, by one freeze-thaw cycle followed by sonication. The lysate was centrifuged at 16 000 g for 30 min at +4°C and loaded onto a Ni-NTA resin. After washing with HBS containing 20 mM imidazole, the protein was eluted with HBS containing 300 mM imidazole. His-tagged TEV protease [Proteins were expressed in protease  was addeFor the purification of dArc1-CL and dArc2-CL, an additional step was required to remove remains of the cleaved MBP tag. This was done by passing the sample through an amylose resin, equilibrated with HBS containing 1 mM EDTA.The Ni-NTA or amylose flow-through was loaded on a Superdex S200 16/600 column, equilibrated with TBS (20 mM Tris-HCl (pH 7.4), 150 mM NaCl). All proteins gave one major peak in the chromatogram. Selected fractions were concentrated using spin concentrators to a final concentration of 10 mg/ml. Protein purity was analyzed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, giving one strong Coomassie-stained band of the expected size. Protein identity was confirmed using mass spectrometry of trypsin-digested in-gel samples, as described .The details of the protein constructs are given in The absolute mass of the proteins was determined by SEC-MALS, using a miniDawn Treos instrument (Wyatt). A Superdex S200 Increase 10/300 equilibrated with TBS was used for sample separation. The system was calibrated using bovine serum albumin, and protein concentration was measured using an online refractometer. Data were analysed with ASTRA (Wyatt).The ellipticity of the proteins was recorded using a Jasco J-810 spectropolarimeter and a 1-mm quartz cuvette. The protein concentration was 0.2 mg/ml in 20 mM phosphate (pH 7). The experiments were done at +20°C.Crystals were obtained by sitting-drop vapor diffusion at +20°C. The crystals of dArc2-NL were grown by mixing 150 nl of dArc2-NL at 8 mg/ml with 150 nl of reservoir solution , 20% PEG 6000). The crystals of dArc2-CL were made by mixing 200 nl of protein at 15 mg/ml with 100 nl of reservoir solution , 200 mM lithium sulphate). The crystals of dArc1-CL were made by mixing 150 nl of the protein at 12 mg/ml with 150 nl of a reservoir, consisting of 100 mM MIB buffer  (pH 5) and 25% PEG 1500. The crystals of dArc1-CL used for phasing were grown by mixing 2 μl of the purified protein at 12 mg/ml with 2 μl of a reservoir solution, consisting of 20% PEG 3350, by hanging-drop vapour diffusion at +20°C. These crystals were soaked in a solution of 20% PEG 3350 with 500 mM NaI for 20 s.Crystals were mounted in loops and snap-cooled in liquid nitrogen. X-ray diffraction data for dArc2-NL were collected on the I03 beamline at Diamond Light Source , while the data for dArc1-CL and dArc2-CL were collected on the P11 beamline  at PETRAab initio models generated by QUARK [via iodine single-wavelength anomalous dispersion (SAD) and the Auto-Rickshaw pipeline [Phasing of dArc2-NL was done with molecular replacement in AMPLE  and ab iby QUARK , using tby QUARK , 30. Thepipeline , with thpipeline , PHASER pipeline , PARROT pipeline , and BUCpipeline . The respipeline , using apipeline . All strpipeline , and modpipeline . The quapipeline . Data prThe PISA server  was usedSynchrotron SAXS data for dArc1-NL and dArc2-NL were collected on the B21 beamline at Diamond Light Source  using a SEC-SAXS setup, where SAXS frames are collected as the sample elutes from a SEC column. A Superdex S200 Increase 3.2 column equilibrated with TBS was used. The injected sample was at 5 mg/ml, and the measurements were done at +10°C. SEC-SAXS data for dArc1-CL and dArc2-CL were similarly collected on the P12 beamline  at EMBL/Ab initio dummy atom and chain-like SAXS models were built with DAMMIN [SAXS data were processed using ATSAS , and theh DAMMIN  and GASBh DAMMIN , respecth DAMMIN  was usedDrosophila and human Arc. Arc in the cell had a concentration of 0.5 mM (dArc N-lobes) or 0.25 mM (hArc N-lobe); the peptide concentration in the syringe was 10-fold higher. The peptide was injected in 26 3-μl injections, with an initial injection of 0.5 μl and a second 0.5-μl injection after 14 injections due to syringe refill. Both the protein and peptide were in TBS buffer. The experiments were done at +25°C, and the data were analyzed with MicroCal Origin 7, using a one-site binding model.A MicroCal iTC200 instrument  was used to determine the binding affinity of a stargazin peptide  to the NL of e-value less than 10−10 [e-values less than 10−20, 10−10, and 10−8 [For conservation calculations, homologues from the non-redundant sequence database were collected using BLAST, accepting hits with an an 10−10 . For larand 10−8 . Full leand 10−8 . Before pi is the frequency of amino acid type i at a given alignment position.Sequence conservation/variability was calculated from the alignments using entropy,Although mArc and dArc both contain similar lobe domains , in mArcThe isolated NL and CL domains of both dArc1 and dArc2 could be produced in soluble form. However, our dArc constructs containing both lobes were poorly soluble; this low solubility resembles that of full-length mArc. While we could obtain pure dArc1 containing both lobes, the protein formed a mixture of large oligomeric states upon storage; dArc2 with both lobes was insoluble in our hands. These problems are likely linked to the missing dArc N-terminal tail in our constructs, which recently was shown to be an integral part of the dArc-NL fold , 22, as The different behaviour of the isolated lobe domains suggests that mArc and dArc differ in the mechanisms, through which they form larger structures, such as capsids. Our aim was to understand the structural basis of these differences, using the individual dArc1 and dArc2 lobe domains for structural studies.2 of buried surface area. The interface consists exclusively of π-π and van der Waals interactions, and the helices encapsulate a hydrophobic core between the monomers, formed by Phe51, Val55, Pro74, Phe77, Ile80, Trp84, Trp95, Leu99, Leu102, and Phe106 . This behaviour of the dArc C-lobes is different to the monomeric mArc C-lobe , while bin vitro [The crystal structures of the dArc C-lobes resemble those of mArc and retroviral capsid proteins . The dimin vitro . Thus, dBoth the intact mArc-CT and the mArc-CL alone are monomeric in solution , 18, 84.Conservation within the Arc-CL  raises sAt the time our experiments were planned and carried out, no data had been published on dArc structure. Since then, both the crystal structure of a dimeric, bilobar dArc1 construct  as well Above, we compared the current structures mainly to the dArc capsids and homologous proteins. Importantly, the crystal structure of dimeric dArc1 , with boet al. [Interesting differences arise, however, when we compare different Arc-NL structures to each other. The dArc1-NL in the structure of Cottee et al.  is monomet al.  clearly et al. [As we were unable to produce dArc constructs with both lobes preset for structural studies, a brief comparison of the protein production methods to recent studies is warranted. Cottee et al.  successfet al. , and capet al. , 22. It The structure and oligomeric state of the dArc lobe domains were analyzed in solution by SEC-MALS, SAXS, and CD , Table 2via interaction of the contrasting surface potentials on each side of the dimer. The lack of this electrostatic polarization in dArc1-NL could be linked to oligomerization . We find 26 sequences with an incomplete NL. More than a third (9) of these are annotated as partial sequences. Of the remaining sequences, none are confirmed to exist, and there is no clear domain boundary in the alignment. Similar results are obtained starting from an NL sequence. This does, however, not prove conclusively that the NL and CL are always found together in eukaryotes, but according to current data, a double-lobed Ty3/gypsy capsid protein is the ancestor of Arc in the animal kingdom.The NL and CL are sequence-related, suggesting a duplication. They are related to viral capsid (Gag) proteins, but the Gag protein in flavi- and other viruses has only one unit, or lobe. One might expect to see either the NL or CL by itself in some cellular organism. A long, iterated search starting from either  domains . DatabasIn eukaryotes from mammals to insects and plants, the overwhelming majority of Arc-like proteins have both NL and CL. This has implications for the Arc evolutionary history. It appears that a functional protein has both lobes, and there are no clear examples of a system with two copies of just an NL or CL. The obvious interpretation is that after a duplication event, the two halves adopted different roles and sites in the two halves have experienced differential evolutionary pressure, as suggested by the conservation plots  and the via two separate integration events of distinct Ty3/gypsy retrotransposons into vertebrates and insects [It was suggested that Arc entered the realm of animals  insects . The earArc is critical to the nervous system, but the protein fold is related to capsid proteins and, at least at the sequence level, even to proteins found in plants. A functional property of the mArc-NL is a peptide binding site, shown to interact with several proteins , 17. ProWe have shown that both lobes of dArc1 and dArc2 are oligomeric in solution. The isolated lobes of the C-terminal domain of mammalian Arc do not exhibit the same propensity for oligomerization . Absent Furthermore, we presented a novel dimeric state of dArc2-NL. This domain-swapped dimer could have a role in the non-capsid functions of dArc. It shares structural similarity with nucleotide- and membrane-interacting proteins, suggesting a related function. How homotetramerization of this lobe, observed in solution, might affect its function, remains to be studied. Overall, the strikingly different behaviour of the purified lobe domains from dArc and mArc points towards different mechanisms in their molecular function and oligomeric assembly. Our data shed light on the individual lobes as small building blocks of dArc capsids, and they complement seminal recent work on dArc structure within capsids  and in iS1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


MicroRNAs (miRNAs) regulate gene expression in a tissue-specific manner. However, little is known about the miRNA expression changes induced by the therapy in rectal cancer (RC) patients. We evaluated miRNA expression levels before and after therapy and identified specific miRNA signatures reflecting disease course and treatment responses of RC patients. First, miRNA expression levels were assessed by next-generation sequencing in two plasma samplings (at the time of diagnosis and a year after) from 20 RC patients. MiR-122-5p and miR-142-5p were classified for subsequent validation in plasma and plasma extracellular vesicles (EVs) on an independent group of RC patients (n=107). Due to the intrinsic high differences in miRNA expression levels between samplings, cancer-free individuals (n=51) were included in the validation phase to determine the baseline expression levels of the selected miRNAs. Expression levels of these miRNAs were significantly different between RC patients and controls . A year after diagnosis, miRNA expression profiles were significantly modified in patients responding to treatment and were no longer different from those measured in cancer-free individuals. On the other hand, patients not responding to therapy maintained low expression levels in their second sampling . Besides, overexpression of miR-122-5p and miR-142-5p in RC cell lines inhibited cell growth and survival. This study provides novel evidence that circulating miR-122-5p and miR-142-5p have a high potential for RC screening and early detection as well as for the assessment of patients’ outcomes and the effectiveness of treatment schedule. MicroRNAs (miRNAs), a class of non-coding RNAs, can modulate gene expression post-transcriptionally by RNA interference . Many stRectal cancer (RC) accounts approximately for 30% of all colorectal cancer. Unlike the tumor in colon, RC tends to be diagnosed at a younger age, it is more frequently associated with lung-only metastasis and requires a different treatment strategy –25. Althin vitro experiments using RC cell lines together with the administration of 5-FU.The present study aimed to describe the changes in miRNA expression in plasma of RC patients before and after therapy and to explain why patients with the same cancer stage may have different treatment susceptibility and long-term outcomes. The expression profiles of selected miRNAs were also compared to those of cancer-free individuals. Identified prognostic miRNAs were further investigated for their involvement in cellular processes  in High throughput sequencing of the whole miRNome was performed on plasma samples from 20 patients with RC (Discovery cohort). Specifically, plasma was sampled at two different time-points: at the time of diagnosis (before surgery) and a year after the diagnosis (corresponding to the time of termination of the therapy). Those miRNAs showing the highest differences in expression levels  between these two samplings, as well as those associated with the therapy response were selected for the validation phase.Significant miRNA expression levels were further validated by real-time quantitative polymerase chain reaction (RT-qPCR) in plasma of an independent group of RC patients  whose sampling was performed in a similar way as the Discovery cohort. Due to the observed high differences in miRNA expression levels between the two samplings, the selected miRNAs were also investigated in a control group consisting of cancer-free individuals (n=51). In addition, for the Validation cohort the expression levels of the selected miRNAs were analyzed also in plasma extracellular vesicles (EVs) in both 2 samplings. For detailed clinical characteristics of patients from both Discovery and Validation cohorts, see in vitro for their effect on cell proliferation, survival, and cell cycle after the administration of 5-FU.Finally, to partially address their mechanism of action, the selected miRNAs associated with the therapy response were further studied A simplified workflow of the study is depicted in The Discovery cohort comprised of 20 patients with RC while the Validation cohort consisted of 107 RC patients and 51 cancer-free volunteers. All patients were recruited between 2007 and 2018 at the Department of Surgery, the Thomayer Hospital in Prague, Czech Republic. Study participants provided information on their lifestyle, body mass index (BMI), and family/personal history of cancer, using a structured questionnaire to determine basic demographic characteristics and potential risk factors for RC. All patients were followed up until August 2020. Clinical data of all patients are represented by clinical stage at diagnosis (classified as the Tumor-Node-Metastasis (TNM) system according to Union for International Cancer Control), grade, information about neoadjuvant and adjuvant treatment regimens, the presence of recurrence, and survival. In both the Discovery and Validation cohorts, RC patients were divided into responders (those who benefit from the chemotherapy and with no relapses) and non-responders .For all patients and controls, peripheral blood was collected into EDTA tubes, stored at 4°C, and centrifugated at 1400 rpm at 4°C for 10 min within 1 hour after its collection for plasma separation. The plasma fraction was immediately frozen and preserved at -80°C.All study individuals signed a written consent to participate in the study and approved the use of their biological samples for genetic analyses according to the Helsinki declaration. The design of the study was approved by the Ethics Committee of the Institute of Experimental Medicine and the Thomayer Hospital, Prague, Czech Republic.RNA from whole plasma was extracted using Plasma/Serum Circulating and Exosomal RNA Purification Kit  according to manufacturer´s protocol.ExoQuick exosome precipitation solution  was used for EVs precipitation from plasma according to manufacturers’ instructions and as described by us . BrieflyFor all samples, RNA concentration was quantified with Qubit 3.0 Fluorometer using the Qubit microRNA assay Kit .The next generation sequencing library preparation for the Discovery set was carried out as described in Sabo et al. . MiRNA lhttp://miarmaseq.cbbio.es/). miARma-Seq is a new comprehensive pipeline analysis suite designed for mRNA, miRNA, and circRNA identification and differential expression analysis, applicable to any sequenced organism. It integrates, among others, the Bowtie tool for read mapping and the miRDeep2 tool for miRNA expression analysis and de novo miRNA prediction , iii) known human miRNA hairpins , and vi) human tRNAs (UCSC database). The alignment to the human tRNA sequences was performed to reveal potential contaminations.All the samples were mapped to the i) reference human genome (hg38 release), ii) known human matured miRNAs  according to the manufacturer´s protocol. RNU48 and RNU6B were used as reference genes selected by Normfinder . For 10 μl reverse transcription reaction, 5 μl of RNA sample (40 ng RNA) were used to prepare cDNA and reaction mixtures were incubated for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C .For evaluating miRNA expression levels in whole plasma and plasma EVs in the Validation cohort, cDNA was synthesized from total RNA by TaqMan MicroRNA Reverse Transcription Kit  and analyzed using TaqMan MicroRNA assays . The pre-amplification reaction contained 2 μl cDNA, 1.5 μl miRNA primer, 5 μl IQ SuperMix and 1.5 μl RNAse free water. Preamplification was performed as follows: 95°C for 3 min, 18 cycles of 95°C for 15 sec and 59°C for 4 min and then held at 4°C . After pre-amplification, each reaction mixture was diluted 100x.RT-qPCR was performed using the Applied Biosystems 7500 Sequence Detection System. The 20 μl PCR reaction mixture consisted of 2 μl of 100x diluted pre-amplified cDNA, 1 μl of primer , 10 μl TaqMan Universal MasterMix II – no UNG  and 7 μl RNAse-free water. Reactions were run in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 10 min.2 in Dulbecco´s Modified Eagle Medium  and in Roswell Park Memorial Institute , respectively, and supplemented with 10% fetal bovine serum , 1 mM L-Glutamine, 100 U/ml penicillin/streptomycin and, in case of RPMI, 1 mM sodium pyruvate (Biosera). Cells were used up to 8 passages.Both RC cell lines (HRA16 and SW1463) were originally obtained from ECACC . The cells were confirmed free from mycoplasma contamination with MycoAlert PLUS Mycoplasma Detection Kit . HRA16 and SW1463 were cultured in a humidified atmosphere at 37°C and 5% CO® RNAiMAX  according to the manufacturer’s protocol. All the experiments were performed in three independent replicates. The transfection efficiency was confirmed by RT-qPCR. After transfection, 5-FU  was dissolved in DMSO  to 5 µM 5-FU, added to selected wells, and cells were treated for 24 hours.Cells were transfected 24 hours after seeding with 10 nM hsa-miR-142-5p  and hsa-miR-122-5p  mimics and miRNA mimics negative control with no homology to the human genome  using LipofectamineThe proliferative capacity in miRNA-modulated cells (750 000 cells/ml) was measured using a water-soluble tetrazolium-1 solution  48 hours after transfection. The WST-1 reagent was added into the media according to the manufacturer´s instructions and measured at 450 and 690 nm. Absorbance was measured using fluorescence reader Biotek ELx808  and proliferation was monitored for different time points . For colony forming assay, transfected cells were plated on 6-well plates (500 cells per well). After 24 hours, cells were treated with 5 μM 5-FU for 24 hours, and then the medium was replaced with fresh medium. After 12 days, colonies were fixed with 3% formaldehyde and stained with 1% crystal violet. The number of colonies was counted manually. Both measurements were performed in triplicates.Cells (750 000 cells/ml) were seeded on 12-well plates (with and without 5-FU treatment), harvested by trypsinization, washed with PBS, and spun down at 1000 rpm at room temperature for 10 min. Then, 1 ml of Propidium iodide (PI) staining solution  was added to the cell pellet and cells were incubated for 30 min at 37°C in the dark. After incubation, samples were measured using flow cytometer Apogee A-50 micro . Measured data were analyzed with Flowlogic software .https://portal.gdc.cancer.gov) using the TCGA biolinks R package . Statistical analysis for TCGA data was performed using the R environment using the dplyr and survival, survminer and ggplot2 package. The survival significance was measured by log-rank test and Mann-Whitney was employed for the differential expression analyses. The significance was set to non-adjusted p = 0.05. The multivariate Cox regression analysis was performed in the R environment using the olsrr package and the ols_step_all_possible function that uses the Akaike information criterion (AIC) criterion for all possible combinations of factors on the patient dataset after screening and EVs and plasma specimen.Differential expression of the miRNA-Seq raw count data was assessed using the EdgeR and Noiseq directly using the miARma-Seq pipeline. Due to the small sample size and the heterogeneity of the RC phenotype, unadjusted p-values were used. Statistical significance was set up at p ≤ 0.05. RT-qPCR statistical analysis was performed by GenEx qPCR data analysis software version 6 (multiD) and R version 3.4.0. The linear mixed models were used (R package lmer and multcomp) for the comparison of miRNA expression in different conditions and different tissues. http://lmmd.ecust.edu.cn/database/smir-nbi/ . miRWalk, an open-source platform, generates up-to-date predicted and validated miRNA-binding sites of known genes [http://mirwalk.umm.uni-heidelberg.de . miR-142-5p was identified as associated with patients’ therapy response: non-responders  exhibited significantly lower expression levels in their second sampling compared to the first one . Downregulation of these miRNAs was calculated comparing the second sampling miR-122-5p and miR-142-5p were validated by RT-qPCR in whole plasma and further in plasma EVs of RC patients (n=107) and cancer-free controls (n=51). Both analyzed miRNAs were detectable in whole plasma and plasma EVs. Results are represented in ;As in the Discovery cohort, in the Validation cohort miR-122-5p was significantly upregulated in the second (T1) plasma sampling when compared with the first one (T0) of RC patients. miR-122-5p expression levels in second sampling (T1) were significantly higher than those in the first sampling .Due to the observed differences between consecutive plasma samplings in RC patients, we wanted to determine the baseline expression levels of selected miRNAs. For this reason, we included plasma samples from a control group consisting of healthy volunteers and compared the miRNA expression levels of RC patients at the first sampling (T0 at the time of the diagnosis) with those from the control group. The results revealed that both, miR-122-5p and miR-142-5p, were significantly down-regulated in RC patients when compared with the control group in plasma samples , the expression levels of both tested miRNAs did not differ between patients that benefited from the therapy and were alive from those dying during the follow-up period , both miRNA expression profiles were significantly modified in responding patients and no longer different from those measured in controls . On the contrary, we observed a significant difference between patients that do not respond a year after diagnosis and the control group .With the advantage of long-term follow-up and gathering the clinical data, we further stratified the patients according to the response to therapy. Identified miRNAs were further evaluated for patient´s clinical characteristics and treatment response . At the time of the diagnosis (1). We did not observe any difference between consecutive plasma EVs samplings. However, after stratification of RC patients based on therapy response, we observed a similar trend as in plasma of RC patients (data not shown). Concerning miR-122-5p EVs expression levels a year after diagnosis (T1), the expression levels in RC patients that benefited from therapy were not different from the expression levels in the control group while patients that did not respond to therapy still evinced lower miR-122-5p expression levels . This trend was not observed for miR-142-5p in plasma EVs . This discrepancy might be caused by a relatively small number of patients that evinced relapse and did not respond well to therapy.EVs are cell-derived membranous structures of endocytic origin and contains various functional protein, mRNA, or miRNAs . DespiteNo significant differences were observed between different stages of RC, neither for miR-122-5p nor for miR-142-5p in EVs and plasma. These non-significant outcomes can be attributed to the small number of patients in some of the stages  on proliferation activity of SW1463 and HRA16 was measured by WST-1 assay. After 24h, a significant decrease in cell proliferation of SW1463 cells after the miR-122-5p transfection was observed . We focused only on those samples included in the TCGA-READ project that had clinical data including survival data, reported miR-122 or miR-142 expression levels. Unfortunately, for miR-122 expression levels were available for 22 patients only and no significant outcomes were noticed (data not shown). Concerning miR-142 expression levels, in non-responders we observed a significantly reduced expression levels for this miRNA when compared to responders  were analyzed using the selection method variables (stepwise selection). This analysis was performed for EVs and plasma samples either considering the expression levels measured in these specimens together or separately.The overall survival for EVs and plasma samples analyzed together was predominantly dependent on pN, sex, pM and Adjuvant chemotherapy (AIC=67.26).By ranking the significance of the combinations of individual factors according to the AIC criterion, the combination `hsa-miR-142`-pN-sex-pM-Adjuvant chemotherapy ranked 9th (AIC=69.18), and the combination` hsa-miR-122`-pN-sex-pM-Adjuvant chemotherapy ranked 11th (AIC=69.22) out of a list 511 possible combinations. These results indicated the potential prediction of survival status of both miRNAs.For the analysis on plasma only, the combination pN and Adjuvant chemotherapy was the strongest predictors (AIC=37.18). Besides, the 8th and 9th place of 255 possible were taken by the combinations containing `hsa-miR-122`-pN-Adjuvant chemotherapy (AIC=39.05) and ` hsa-miR-142`-pN-Adjuvant chemotherapy (AIC=39.16), respectively.Considering EVs only, the outcomes were similar to those obtained in plasma. The most significant results were for the parameters pN and Adjuvant chemotherapy (AIC=40.89) while the combination `hsa-miR-122`-pN-Adjuvant chemotherapy (AIC=42.09) and` hsa-miR-142`-pN-Adjuvant chemotherapy (AIC=42.85) were on the 3rd and 7th position, respectively.In summary, the majority of tested models (approx. 70%) included miRNAs as significant predictors (data not shown).in silico analysis, both miRNAs resulted regulated by many drugs, such as cisplatin or 5-FU. This in silico outcome agrees with our findings that both RC cell lines with artificially increased miRNA levels had worse survival after 5-FU therapy.To identify whether both tested miRNAs could work as potential pharmacogenomic biomarkers for anticancer drugs, we have performed an additional analysis with the implementation of SMiR-NBI model which predicts interactions between small molecules and miRNAs  included in miRWalk. Target genes that could be predicted in at least three databases were defined as highly predicted miRNA targets. For these target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted by GSEA.For miR-122-5p, 236 target genes were predicted according to miRWalk and validated in miRTarBase  were selected for further validation. To investigate whether these miRNAs might be used as non-invasive diagnostic and prognostic biomarkers, miR-122-5p and miR-142-5p were analyzed in plasma and further in plasma EVs of 107 RC patients and 51 cancer-free individuals. Plasma and plasma EVs of RC patients were examined at the time of diagnosis and one year after. EVs are considered as both mediators of cell-to-cell communication, and as potential delivery vesicles for a therapeutic approach , 37. We To further explain our observation, both miRNAs were artificially overexpressed in RC cell lines (SW1463 and HRA16) and investigated for their effect on long-term survival with and without administration of 5-FU. Increased levels of both miRNAs in RC cell lines were associated with significantly less formed colonies after administration of 5-FU. These results are in concordance with RC patients and suggest that miR-122-5p and miR-142-5p act as tumor suppressors. In addition, it has been proved that miR-122-5p is up-regulated in the presence of 5-FU by SMiR-NBI model and it might be assumed that the miRNA effect is thus potentiated. Studies connected to colon and rectal pathogenesis and miR-122-5p expression levels were mainly focused on metastatic colorectal cancer. One of the most frequent sites for colon and rectal cancer metastasis is liver and miR-122-5p was identified as liver-specific miRNA –42. SeveTo the best of our knowledge, the study of Kunigenas et al. is the only one focused on miR-142-5p in RC. The authors reported increased expression levels of this miRNA in rectal tissue compared to adjacent non-malignant mucosa . In coloThe major advantage of our study is specific focus on RC patients. Moreover, all samples were enrolled in a single-center hospital and were of the same ethnicity and socio-cultural background. Plasma samples, as the source for miRNA expression analysis, were collected repeatedly within a year with the ability to monitor the therapy outcome. Generally, most studies are focused on the identification of diagnostic or prognostic biomarkers in body fluids collected only at the time of the diagnosis. The consecutive blood samplings as performed in the present study are usually lacking and this highlight the unique design of our study. In comparison with a traditional tissue biopsy, plasma samples bring advantages of easy, non-invasive collection that can be repeatedly collected overtime. The main limitation of this study is a relatively small cohort of patients with an available second sample and a lack of tissue material. The present study is, to our knowledge, the first study to evaluate changes in miRNA expression levels within the RC patients over the course of treatment, and their correlation with their follow-up.https://www.ncbi.nlm.nih.gov/bioproject/727696.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Trace Archive NCBI Sequence Read archive The studies involving human participants were reviewed and approved by Committee of the Institute of Experimental Medicine and the Thomayer Hospital, Prague. The patients/participants provided their written informed consent to participate in this study.in vitro analysis – AO. Statistical analysis – VN. Interpretation of data – BP, AN, and VV. Original draft preparation – KC. Writing – reviewing and editing – VV, BP, AN, PV, KC, TB, SV, ML, AO, PS, VN and JH. All authors contributed to the article and approved the submitted version.Design study and performance of discovery phase – VV, BP, and AN. Collection and preparation of samples – KC, ML, PS, and TB. Performance of validation phase – KC. Supervision This project was supported by the Grant Agency of the Ministry of Health of the Czech Republic (AZV 17–30920A) and the Grant Agency of Charles University (GAUK 302119). The work was partially supported by LegaI taliana per La Lotta contro i Tumori (LILT) (to BP and AN) and by the Oncobiome European H2020 research project (grant number 825410 to AN).TB received honoraria and research support from Roche, Bayer, Ipsen, Novartis, Merck, Bristol-Myers Squibb, and Servier (unrelated to the present article).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.